Molecular diagnostics of some trichodorid nematodes and associated Tobacco rattle virus

Abstract

Several Paratrichodorus and Trichodorus (trichodorid) nematode species are natural vectors of Tobacco rattle virus (TRV) and cause economically important diseases, especially in potato and ornamental bulbous crops. Identification of trichodorid species based on morphological characters is laborious, time-consuming, and requires the services of highly trained personnel. Molecular diagnostics for trichodorid nematodes, using the ribosomal DNA repeat unit, were successfully developed to distinguish two Paratrichodorus and two Trichodorus species. The complete sequences of the 18S genes and the ITS-1 regions for these species were obtained and species-specific primers successfully designed for them. An RT-PCR assay was developed utilizing isolate-specific primers that amplify serologically distinguishable strains of TRV in individual trichodorid nematodes. The primers were based on the highly conserved RNA-1 segment of the bipartite genome and also on different parts of the RNA-2 segment of the virus genome.

Molecular characterization and diagnostics of stubby root and virus vector nematodes of the family Trichodoridae (Nematoda: Triplonchida) using ribosomal RNA genes

“…Recently, DNA-based approaches have been successfully adapted for the molecular diagnostics of trichodorids. Blaxter et al (1998), Boutsika et al (2004b), Riga et al (2007), Van Megen et al (2009) and Duarte et al (2010) have published sequences of 18S rRNA and ITS rRNA genes for several agriculturally important species of trichodorids. Using sequence data, Boutsika et al (2004a) and Riga et al (2007) developed a PCR with specific primers for diagnostics of P. allius, P. macrostylus, P. pachydermus, P. teres, T. primitivus and T. similis.…”

First report of Trichodorus primitivus, T. sparsus and T. viruliferus (Nematoda: Trichodoridae) from the Czech Republic

“…Systematic research on Trichodorus is time consuming and novel techniques that will facilitate detection of Trichodorus in a sensitive and specific way are increasingly being sought. New methodologies employing polymerase chain reaction and DNA sequencing has been reported (Boutsika et al, 2004a;Boutsika et al, 2004b). In these studies PCR with a species-specific differentiation constitutes a major step forward in developing DNA diagnostics.…”

“…Specimens for morphometric and morphological analysis from the field samples were extracted by sieving over 1mm, 150 µm and 75µm and placing the residual on a tissue paper on a Baermann funnel from 24 -48 hours (Brown & Boag, 1988). Nematodes were heat killed, fixed in TAF, processed in slow glycerin process and mounted in anhydrous glycerin on slides.…”

An overview on therapeutic potential and various applications of microbial collagenases

“…Thus, to detect plant viruses carried internally by these nematodes the nematode cuticle must be disrupted by rather severe means. Researchers have disrupted nematode cuticles mechanically by microdissection [169], or by vortexing in the presence of phenol and glass beads [170]. These methods were evaluated for the detection of Tomato ringspot virus [ToRSV] in Xiphinema americanum, transmitted to healthy plants by viruliferous nematodes in the soil.…”